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[Bio101] Lifespan Assay
[Bio101] 用5-氟脱氧尿(FUDR)苷测定寿命   

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Abstract

This assay is used to address aging-related questions in worms.

Materials and Reagents

  1. NGM medium
  2. 5-fluorodeoxyuridine (FUDR) (Sigma-Aldrich, catalog number: F0503 )

Equipment

  1. Incubators
  2. NGM agar plates (35 x 10 mm)
  3. 60 x 15 and 35 x 10 mm petri dish (VWR)

Procedure

  1. Synchronize worms by either egg prep or egg lay.
  2. Animals are grown on NGM plates until they reach the L4 stage (about 36 h at 25 °C).
  3. Make small NGM agar plates (35 x 10 mm) containing 5-fluorodeoxyuridine (FUDR; 0.1 mg ml-1) to prevent growth of progeny. Make plates fresh and store in the dark at 4 °C up to one week.
  4. Seed a FUDR-containing plate fresh. Spot overnight OP50-1 culture (50 μl of 20x concentrated culture) onto FUDR plates one day before transferring worms. Keep seeded plates at RT overnight.
  5. Transfer 30 L4 to each FUDR plate and 4-5 plates for each strain.
  6. Grow at desired temperature (15, 20, or 25 °C). Animals are scored every 1 to 3 days subsequently and scored as dead when they no longer respond to gentle prodding with a platinum wire. Remove the dead worms after counting. Worms found dehydrated on plate walls are not counted as dead worms.
  7. Lifespan is defined as the day animals are at the L4 larval stage (time t = 0) until the day they are scored as dead.

References

  1. Wolkow, C. A., Kimura, K. D., Lee, M. S. and Ruvkun, G. (2000). Regulation of C. elegans life-span by insulinlike signaling in the nervous system. Science 290(5489): 147-150.
  2. Apfeld, J. and Kenyon, C. (1999). Regulation of lifespan by sensory perception in Caenorhabditis elegans. Nature 402(6763): 804-809.
  3. Mitchell, D. H., Stiles, J. W., Santelli, J. and Sanadi, D. R. (1979). Synchronous growth and aging of Caenorhabditis elegans in the presence of fluorodeoxyuridine. J Gerontol 34(1): 28-36.

简介

该测定用于解决蠕虫中与老化相关的问题。

材料和试剂

  1. NGM媒体
  2. 5-氟脱氧尿苷(FUDR)(Sigma-Aldrich,目录号:F0503)

设备

  1. 孵化器
  2. NGM琼脂平板(35×10mm)
  3. 60×15和35×10mm培养皿(VWR)

程序

  1. 通过准备蛋或同步蛋同步蠕虫。
  2. 动物生长在 NGM板上,直到它们达到L4阶段 在25℃下约36小时)
  3. 制备含有5-氟脱氧尿苷(FUDR; 0.1mg ml -1 -1)的小NGM琼脂平板(35×10mm)以防止子代的生长。 使板新鲜和存储在黑暗中4°C长达一个星期。
  4. 新鲜种植含FUDR的板。 在转移蠕虫前一天,将过夜OP50-1培养物(50μl的20倍浓缩培养物)点在FUDR板上。 将接种的平板在室温下保持过夜
  5. 转移30 L4到每个FUDR板和4-5板的每个应变
  6. 在所需温度(15,20或25℃)下生长。 随后每1至3天对动物进行评分,当他们不再用铂丝温和刺激时,评分为死亡。 计数后清除死亡的蠕虫。 发现板壁上脱水的蠕虫不算作死亡蠕虫。
  7. 寿命被定义为日动物处于L4幼虫阶段(时间t = 0)直到它们被评分为死亡的那天。

参考文献

  1. Wolkow,C.A.,Kimura,K.D.,Lee,M.S.and Ruvkun,G。(2000)。 通过胰岛素样信号传导在神经系统中调节秀丽隐杆线虫的寿命。 em> Science 290(5489):147-150。
  2. Apfeld,J。和Kenyon,C。(1999)。 通过Caenorhabditis elegans中的感觉知觉来调节寿命。 自然 402(6763):804-809
  3. Mitchell,D.H.,Stiles,J.W.,Santelli,J。和Sanadi,D.R。(1979)。 在氟脱氧尿苷存在下,秀丽隐杆线虫的同步生长和衰老。 Gerontol 34(1):28-36
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). Lifespan Assay . Bio-protocol Bio101: e57. DOI: 10.21769/BioProtoc.57;
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Ruisheng LU
ENS LYON
So any idea of life span measuring without FuDR?
6/4/2013 9:13:43 AM Reply
Peichuan Zhang
Calico Life Sciences
Per request of Dr. Fanglian He, I will continue to add more comments/tips to this lifespan protocol --- a self-improvement process of our website to provide better protocols.

In the lab we have followed, basically, a protocol that was described in a previous paper (http://www.ncbi.nlm.nih.gov/pubmed/9790527 Apfeld, J and Kenyon, C. 1998. Cell nonautonomy of C. elegans daf-2 function in the regulation of diapause and life span. Cell. 95:199-210.)

The usage of FuDR has been a concern to some researchers. For example, Hekimi's group from Canada has shown that FuDR can extend lifespan of mitochondria mutant gas-1 by two-fold (Van Raamsdonk, J and Hekimi, S. 2011. FUdR causes a twofold increase in the lifespan of the mitochondrial mutant gas-1. Mech Ageing Dev. 132:519-21). In my hand, I often found wild type live a bit longer on plates with FuDR (e.g., ~19 days of mean lifespan @ 20C, no FuDR; ~20 days or even longer, with FuDR). I found that FuDR cannot be used for certain assays, such as worm paralysis assay (e.g., CL2006 abeta strain, generated by Chris Link from Colorado, in which human abeta1-42 is overexpressed in body wall muscles and causes age-associated paralysis).

To a previous question about liquid lifespan assays, please refer to another online protocol (Solis, G and Petrascheck, M. 2012. Measuring Caenorhabditis elegans life span in 96 well microtiter plates. J Vis Exp. 48). This is really a nice protocol from the Petrascheck group at Scripps. Petrascheck published the 2007 Nature paper and demonstrated that serotonin antagonists extend lifespan of worms.
7/8/2012 3:37:19 PM Reply
how we can add the drug or plant extract to the NGM plates?
4/18/2012 12:21:36 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hello,

To address the question that you have raised here, I should first say that I only have very limited experience with assays using small molecules. I'd be very glad to share the experience with you though.

Typically, I'd add the extracts/small molecules onto the plate to a final concentration of 50uM to 100uM. The cuticles of worms, as well as the chemical properties of the small molecules, have very strong impact on the final bioavailability of the chemical agents that you will test. I'd say that 100uM is probably a good start point, as the final concentration inside the worms could be less than 1uM. You may have to test a range of doses though. I'm not sure what kind of assay you will be doing. In the case of lifespan assays, it'd be better to start the treatment at adult stage to avoid potential effects during development.

You can add the extracts/small molecules directly onto NGM plates, or supplement it to the medium. Make sure that high temperature won't cause issues of degradation/oxidation, etc. Try to prepare and use fresh plates, and cover plates with aluminum foil to protect them from light --- it should be OK to store them at 4C for short term (high level of DMSO may form crystals though).

I'd prefer to use salts to get better solubility, and particularly, for hydrophobic stuff such as lipids. Tergitol (Sigma, 0.1% final) can be used to help dissolve lipids on plate as well. Should DMSO be used as the solvent, DMSO only should be added to the control plates as well. Try to control the final level of DMSO to be less than 0.5%. In this case, it'd be better to prepare a stock solution of more than 100mM (DMSO final concentration would be 0.1% after 1000X dilution).

A 2010 BBRC paper from a group in China showed that DMSO (0.5% to 2.0% final) actually extends lifespan in a sir-2.1- and daf-16-dependent manner.
Lifespan extension in Caenorhabditis elegans by DMSO is dependent on sir-2.1 and daf-16.
http://www.ncbi.nlm.nih.gov/pubmed/20828537


Here is some good references that would be helpful to your question:
1. 2006 Aging Cell paper from Cathy Wolkow's lab at NIA/NIH
Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans.
http://www.ncbi.nlm.nih.gov/pubmed/16441844

2. 2007 Nature paper from Linda Buck' lab at Fred Hutchinson
An antidepressant that extends lifespan in adult Caenorhabditis elegans.
http://www.ncbi.nlm.nih.gov/pubmed/18033297

2009 Ann N Y Acad Sci from the same lab about screen
A high-throughput screen for chemicals that increase the lifespan of Caenorhabditis elegans.


3. 2010 PLoS One paper from a German group
Antidepressants of the serotonin-antagonist type increase body fat and decrease lifespan of adult Caenorhabditis elegans.
http://www.ncbi.nlm.nih.gov/pubmed/19112515

Please note that this group reached a conclusion that is exactly the opposite to what the Buck lab found for the effects of serotonin antagonists on lifespan. The major reason, they believe, could be that they used liquid instead of solid plate.

4. 2010 PLoS One paper from Monica Driscoll's lab at Rutgers
Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1.
http://www.ncbi.nlm.nih.gov/pubmed/20090912

5. 2010 Nat Chem Biol paper from Peter Roy's group in Canada
A predictive model for drug bioaccumulation and bioactivity in Caenorhabditis elegans.
http://www.ncbi.nlm.nih.gov/pubmed/20512140

Best,
Peichuan

4/18/2012 1:51:13 PM